Transdermal Pharmaceutical Delivery Composition

ABSTRACT

A pharmaceutically delivery system is described comprising a pharmaceutically active agent and acidified nitrite as an agent to produce local production of nitric oxide at the skin surface. The dosage form may be in any pharmaceutically acceptable carrier means and comprises an acidifying agent adapted to reduce the pH at the environment. In one embodiment, a barrier consisting of a membrane allows diffusions of the anaesthetic and nitrite ions while preventing direct contact of the skin and acidifying agent.

The present invention relates to a new composition for transdermaldelivery of topically applied pharmaceutical preparations. The systemcomprises the use of the pharmaceutical agent and acidified nitritecontained within a delivery system to allow passage of both the specificpharmaceutical agent and nitric oxide to the skin.

The penetration of substances through the skin is important from bothtoxicological and therapeutic viewpoints. Passive delivery of mostcompounds across different epithelia is limited due to the excellentbarrier properties afforded by these epithelia. The stratum corneum isthe principal barrier to penetration of most chemicals. Conventionaltopical delivery systems are therefore restricted to either substancesfor local effects or to highly potent, small, lipophilic substances forsystemic effects. It is also difficult to deliver ionic andhigh-molecular-weight drugs in therapeutically sufficient amounts byconventional systems.

By way of example, many medical and surgical procedures require topicalanaesthesia. The use of local anaesthetics requires an agent possessingthe following general properties. It should not be irritating to thetissue to which it is applied, nor should it cause any permanent damageto nerve structure. Its systemic toxicity should be low because it iseventually absorbed from its site of administration. It is usuallyimportant that the time required for the onset of anaesthesia should beas short as possible. Furthermore, the action must last long enough toallow time for the contemplated medical or surgical intervention, yetnot so long as to entail an extended period of recovery (J. MurdochRitchie & N. M. Greene Local Anaesthetics in Goodman & Gilman's: ThePharmacological Basis of Therapeutics, pages 311-331, McGraw-Hill Inc,(1992)).

Local anaesthetics are rapidly absorbed into the circulation followingtopical administration to mucous membranes or denuded skin. It isextremely useful in achieving loss of sensation in a subject without theloss of consciousness, or the impairment of central control of vitalfunctions. Typical uses, include minor invasive procedures such asvenepuncture, e.g. for the collection of blood for diagnostic purposesfrom a patient, for the administration of therapeutic agents, wholeblood or blood plasma to a patient, or prior to the administration of ageneral anaesthetic to a patient. However, it is a common feature amongpatients that the pain of injection can cause discomfort and in certaincases a patient, in particular juvenile subjects, can experience acuteanxiety or panic brought on by the sight of a needle or of the injectionitself. Such panic attacks can be characterised by fainting, vomiting orother related symptoms. Whether the adverse reaction is pain or a panicattack, the problem leads to poor patient compliance with advisablemedical procedures. There exists a need therefore for improved localanaesthetic compositions that can overcome these problems.

Intact, healthy human skin presents an excellent natural barrier to theexternal environment and restricts the passive diffusion ofpharmaceuticals. Local anaesthetics do not readily penetrate intact skin(McCafferty et al Br J Anaesth 60, pages 64-69 (1988)).

The insertion of a needle through the skin, for procedures such asphlebotomy or vaccination, is painful and may induce great fear andanxiety especially in children and the elderly. Painful experiences leadto reduced compliance, with heightened anticipatory anxiety and fear.The introduction of topically-applied cutaneous anaesthetic preparationssuch as EMLA™ (Eutectic Mixture of Local Anaesthetics) cream [AstraPharmaceuticals Ltd.] (Arts et al Pediatrics 93, pages 797-801 (1994))and more recently Ametop Gel™ [Smith & Nephew Healthcare Ltd.] (Freemanet al Paediatr Anaesth 3, pages 129-138 (1993)), represented a definiteadvance in clinical practice and are contributing to breaking the cycleof “needle phobia”.

Studies of the effects of these preparations have produced variableresults (Molodecka et al Br J Anaesth 72, pages 174-176 (1994); Lawsonet al Br J Anaesth 75, pages 282-285 (1995)). However, relatively slowonset times (EMLA™ 60-90 minutes; Ametop Gel™ 30-45 minutes) remain adeterrent to widespread clinical and patient acceptance with the need toorganise clinic, ward and operating theatre routines accordingly. Thesemethods are of no benefit in acute situations. Additionally, evenfollowing the manufacturer's recommendations for dosage andadministration, potential exists for improvement in the degree ofanaesthesia afforded by these treatments.

A percutaneous local anaesthetic with a more rapid onset time andincreased potency would be helpful in organisational terms for emergencycases, community medicine and for an increasing number of paediatricmedical and surgical day cases. Shortening of the period of anticipatoryanxiety while achieving the maximal desensitising of the skin wouldclearly be clinically advantageous.

Nitric oxide [NO] is a potent vasodilator synthesised and released byvascular endothelial cells and plays an important role in regulatingvascular local resistance and blood flow (Palmer et al Nature 327, pages524-6 (1987)). In mammalian cells, NO is produced along withL-citrulline by the enzymatic oxidation of L-arginine. Nitric oxide isalso involved in the inhibition of both platelet and leukocyteaggregation and adhesion, the inhibition of cell proliferation, thescavenging of superoxide radicals and the modulation of endotheliallayer permeability. Nitric oxide also has been shown to possessanti-microbial properties, reviewed by F. C. Fang (1997) (J. Clin.Invest. 99 (12) 2818-2825 (1997)).

A potential therapeutic utility of the anti-microbial properties of NOis described in WO 95/22335. A pharmaceutical composition comprisingnitrite in an inert carrier cream or ointment and salicylic acid wasused to show killing of cultures containing E. coli and C. albicans.This activity was further tested against patients with fungal infectionof the feet (“Athlete's Foot” or Tidea pedis) and showed that thecondition was amenable to treatment with the acidified nitritecomposition. However, the composition of nitrite and organic acid causederythema (redness) of the skin.

In addition to internal cell-mediated production, NO is also continuallyreleased externally from the surface of the skin by a mechanism, whichappears to be independent of NO synthase enzyme. Nitrate excreted insweat is reduced to nitrite by an unknown mechanism, which may involvenitrite reductase enzymes, which are expressed by skin commensalbacteria. Alternatively mammalian nitrite reductase enzymes may bepresent in the skin which could reduce nitrite rapidly to NO on the skinsurface (Weller et al J Invest Dermatol 107, pages 327-331 (1996)).

The production of NO from nitrite is believed to be through thefollowing mechanism:

NO₂ ⁻+H⁺

HNO₂  [1]

2HNO₂

N₂O₃+H₂O  [2]

N₂O₃

NO+NO₂  [3]

Topical application of a sodium nitrite/ascorbic acid NO-generatingsystem causes significant increases in skin blood flow in patients withRaynaud's disease and in normal healthy subjects without causing localirritation (Tucker et al Lancet 354(9191):1670-5 (1999); Harwick et alClinical Science 100, pages 395-400 (2001)). The reaction can beterminated within a few seconds by gentle wiping of the skin with atissue.

It has now been found that an improved delivery system forpharmaceutically active agents by topical application to the skin can beprepared from a suitable drug and a source of nitrite ions in an inertcarrier cream or ointment when mixed with an organic acid such asascorbic acid. The source of nitrite ions and the organic acid react toproduce oxides of nitrogen to cause sustained vasodilation of themicrocirculatory blood vessels, without significant inflammation. Thisnew use for acidified compositions containing nitrite is a departurefrom the previously known uses of the composition as an anti-microbialagent. The side-effects of erythema associated with the treatment offungal infections of the foot had been considered to suggest that thecomposition should not be used on broken skin or away from sites ofinfection needing immediate, short term therapeutic treatment.Additionally, the skin on the foot is significantly thicker and tougherthan elsewhere on the mammalian body and so can endure more prolongederythema than other thinner areas of skin elsewhere. Furthermore, thereis a widespread and generally accepted medical prejudice againstinserting ointments or gels into open wounds or onto broken skin. Suchpractice is advised against because of the risk of actually causinginfection or blood poisoning. The administration of a pharmaceutical orpharmaceuticals using this system has advantages over previous modes ofadministration.

This system overcomes the limitations associated with conventionaltransdermal pharmaceutical application and is feasible for ionisable,hydrophilic and higher molecular weight compounds. The pharmaceuticalsenter the skin through intracellular spaces and specialised tissues suchas eccrine and apocrine sweat ducts and hair follicles with sebaceousglands.

The system depends upon several variables in addition to factors, whichaffect the skin uptake of drugs during passive diffusion. These includevehicle pH, ionic strength, transport number of ions and water, drugconductivity, solute concentration and skin impedance.

With reference to systemically active compounds, trans-dermal deliveryhas several advantages, particularly avoidance of gastrointestinalincomparability and hepatic “first-pass” effect. Additionally, thenitric oxide induced vasodilation of the skin microcirculationsignificantly enhances percutaneous absorption of the pharmaceuticalagent in to the systemic circulation.

According to a first aspect of the invention there is provided acomposition comprising a pharmaceutically active agent, apharmacologically acceptable acidifying agent, a pharmacologicallyacceptable source of nitrite ions or a nitrite precursor therefore.

The pharmaceutically active agent may comprise any suitable drug orcombination of drugs to treat a disease in a patient. The agent may beimmediately active in the form administered or may become active in thebody of the patient following administration, such as for examplethrough hydrolysis or by the action of an endogenous enzyme. Inprinciple, any pharmaceutically active substance can be administeredusing this delivery system.

Therapeutically, the novel system facilitates the delivery of a widenumber of systemically active substances. Active substances include, butare not limited to, antibiotics, hormones, proteins, peptides,proteogylcans, nucleotides, oligonucleotides (such as DNA, RNA, etc.),vitamins, minerals, growth factors, non-steroidal anti-inflammatorydrugs (NSAIDs). In a preferred embodiment, the delivery system of thepresent invention can be used to deliver anaesthetic, analgesic,hormone, immunosuppressant or steroid formulations. Other pharmaceuticalagents include, but are not limited to, analgesic agents such asibuprofen, indomethacin, diclofenac, acetylsalicylic acid, paracetamol,propranolol, metoprolol, oxycodone, thyroid releasing hormone, sexhormones such as oestrogen, progesterone and testosterone, insulin,verapamil, vasopressin, hydrocortisone, scopolamine, nitro-glycerine,Isosorbide dinitrate, anti-histamines (such as terfenadine), clonidineand nicotine, immunosuppressant drugs (such as cyclosporin), steroids.

The anaesthetic can be any appropriate anaesthetic for local anaesthesiaand can be provided in aqueous or powdered form, for example,amethocaine (tetracaine), lignocaine (lidocaine), xylocalne,bupivacaine, prilocaine, ropivacaine, benzocaine, mepivocaine orcocaine, or a mixture thereof, preferably in the hydrochloride form. Thegeneral concentration range is around 1 to 4% w/w, although greater orlesser amounts can be empirically determined by a physician. Suitablypreferred concentrations are tetracaine (0.01 to 10% w/w, suitably 1 to8% w/w, preferably 2% w/w), lidocaine (0.01 to 10% w/w, suitably 1 to 8%w/w, preferably 5% w/w) and cocaine (1 to 4% w/w). Generally acceptedsafe dosages of such compounds for topical anaesthesia in a healthy 70kg-adult are 750 mg for lidocaine, 200 mg for cocaine, and 50 mg fortetracaine. Other suitable anaesthetics are within the competence of themedical practitioner and can also be used in the composition of thepresent invention at the relevant concentrations.

Prior art methods of improving local anaesthesia have suggested the useof low concentrations of vasoconstrictors, such as phenylephrine(0.005%). However, the compositions of the present invention utilise apreviously unknown property of an acidified nitrite composition toproduce NO, a vasodilator, which accelerates the transfer of anaestheticinto the dermis. The combination of the NO-generating system andanaesthetic will promote patient compliance of venepuncture andblood-letting techniques by reducing the pain experienced during theprocedure.

The choice of pharmaceutically active agent may be determined by itssuitability for the treatment regimen of the disease or medicalcondition concerned and reference can be made to standard referenceworks such as Martindale, the Merck Index, Goodman & Gilman's “Thepharmacological basis of therapeutics”, eighth edition (1992), McGrawHill.

The pharmacologically acceptable acidifying agent is adapted to reducethe pH at the site of application and can include any suitable organicacid. For example, the organic acid can be a (C₁-C₆) alkyl carboxylicacid, a (C₆-C₁₀) aryl (C₁-C₆) carboxylic acid.

As used herein, the term “(C₁-C₆) alkyl” refers to straight chain orbranched chain hydrocarbon groups having from one to six carbon atoms.Illustrative of such alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl. Theterm “(C₆-C₁₀) aryl” includes phenyl and naphthyl.

According to Martindale The Extra Pharmacopoeia, 28^(th) edition (1982),pharmaceutically acceptable acidifying agents can include: dilutehydrochloric acid, betaine hydrochloride, acetic acid, citric acid,citric acid monohydrate, fumaric acid, lactic acid, maleic acid, malicacid, tartaric acid

Other acceptable acidifying agents, include but are not limited to,hexose or pentose sugar molecules substituted with a (C₁-C₆) carboxylgroup, or furanolactone or pyranolactone molecules substituted with a(C₁-C₆) carboxyl group.

Preferred acidifying agents, include, but are not limited to, ascorbicacid (vitamin C), salicylic acid, acetyl salicylic acid, a (C₁-C₆) alkylcarboxylic acid, for example ethanoic acid (acetic acid), citric acid,or a salt, or a derivative thereof in a concentration up to 20% w/w,suitably 0.25 to 10% w/w, preferably 4 to 6% w/w. A particularlypreferred concentration is 4% or 5% w/w. The preferred pH range is frompH2 to pH7, preferably pH4. Other acidifying agents include but are notlimited to, ammonium or aluminium salts, (C₆-C₁₀) aryl compounds such asphenol, benzoic acid or derivatives thereof. Inorganic acids such ashydrochloric acid may be used if sufficient dilute and/or appropriatelybuffered. The acidifying agent may be present as a dissolved salt or ina liquid form.

The pharmacologically acceptable source of nitrite ions may an alkalinemetal nitrite or an alkaline earth metal nitrite. For example, LiNO₂,NaNO₂, KNO₂, RbNO₂, CsNO₂, FrNO₂, Be(NO₂)₂, Mg(NO₂)₂, Ca(NO₂)₂,Sr(NO₂)₂, Ba(NO₂)₂, or Ra(NO₂)₂. Alternatively, a nitrite precursor maybe used as the source of the nitrite ions in the composition, such asfor example a dilute solution of nitrous acid. Other sources of nitriteions are nitrate ions derived from alkali metal or alkaline earth metalsalts capable of enzymic conversion to nitrite. For example, LiNO₃,NaNO₃, KNO₃, RbNO₃, CsNO₃, FrNO₃, Be(NO₃)₂, Mg(NO₃)₂, Ca(NO₃)₂,Sr(NO₃)₂, Ba(NO₃)₂, or Ra(NO₃)₂. The concentration of the nitrate ionsource may be up to 20% w/w, suitably 0.25 to 10%, preferably 4 to 6%. Aparticularly preferred concentration is 4% or 5% w/w.

Suitably, the final nitrite ion concentration present in the compositionis up to 20% w/w, generally in the range of from 0.25% to 15% w/w,suitably 2% to 10% w/w, preferably 4 to 6% w/w. A particularly preferrednitrite ion concentration is 4% or 5% w/w.

In the preparation of an agent according to this aspect of theinvention, the pharmaceutically active agent, the acidifying agent andthe nitrite ions or source therefore are formulated in apharmacologically acceptable carrier or diluent which may be an inertcream or ointment. In a particular preferred form of the invention thepharmaceutically active agent, the acidifying agent and the source ofnitrite ions or precursor therefore are separately disposed in the saidcream or ointment for admixture to release ions at the environment ofuse.

The pharmaceutical composition may be adapted for administration by anyappropriate topical route, including buccal, sublingual or transdermal.Such compositions may be prepared by any method known in the art ofpharmacy, for example by admixing the active ingredient with thecarrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research,3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. For treatment of theeye or other external tissues, for example mouth and skin, thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active ingredient may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredient may be formulated in a cream with an oil-in-watercream base or a water-in-oil base. Pharmaceutical compositions adaptedfor topical administration to the eye include eye drops wherein theactive ingredient is dissolved or suspended in a suitable carrier,especially an aqueous solvent. Pharmaceutical compositions adapted fortopical administration in the mouth include lozenges, pastilles andmouth washes.

The pharmaceutical compositions may contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colourants, odourants, salts (substances of the presentinvention may themselves be provided in the form of a pharmaceuticallyacceptable salt), buffers, coating agents or antioxidants. They may alsocontain therapeutically active agents in addition to the substance ofthe present invention.

Dosages of the substance of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used.

This dosage may be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be reduced, inaccordance with normal clinical practice.

Such compositions may be formulated for human or for veterinarymedicine. The present application should be interpreted as applyingequally to humans as well as to animals, unless the context clearlyimplies otherwise.

According to a second aspect of the invention there is provide the useof a composition as defined above in medicine. In a third aspect of theinvention, there is provided the use of a pharmaceutically active agent,a pharmacologically acceptable acidifying agent, a pharmacologicallyacceptable source of nitrite ions or a nitrite precursor therefore inthe preparation of an agent for the treatment of a disease or medicalcondition. The medical condition can include local anaesthesia,immunosuppression, e.g. to prevent transplant rejection. Diseasessuitable for treatment using the delivery system of the presentinvention, include but are not limited to cardio-vascular diseases,neurological diseases or disease of the central nervous system, (e.g.multiple sclerosis, Parkinsons' Disease), epilepsy, psychiatricdisorders (e.g. schizophrenia), inflammation (e.g. rheumatoid arthritis,osteoarthritis, asthma, gout), in particular topical inflammation,hypertension, arrhythmia, hyperlipoproteinemias, gastrointestinaldisorders (e.g. peptic ulcers), kidney disease, parasite infections(e.g. protozoal infection, helminthiasis, amebiasis, giardiasis,thichomoniasis, leishmaniasis, trypanosomiasis, malaria), microbialinfection (e.g. yeast, fungus, bacteria), viral infection, cancer,immunosuppression, blood disorders (blood clots etc.), endocrine (e.g.hormonal) disorders (e.g. thyroid condition, hypoglycaemia), diabetes,dermatological disorders (e.g. psoriasis).

According to a fourth aspect of the invention there is provided a methodfor the local anaesthesia of the skin of a patient, comprising theadministration of a composition comprising a anaesthetic, apharmacologically acceptable acidifying agent, a pharmacologicallyacceptable source of nitrite ions or a nitrite precursor therefore.

According to a fifth aspect of the invention there is provided acomposition comprising an pharmaceutically active agent, apharmacologically acceptable acidifying agent, a pharmacologicallyacceptable source of nitrite ions or a nitrite precursor therefore as acombined preparation for simultaneous, separate or sequential use intreatment of a disease or medical condition defined above.

According to a sixth aspect of the invention there is provided a kitcomprising a pharmaceutically active agent, a pharmacologicallyacceptable acidifying agent and a pharmacologically acceptable source ofnitrite ions or a nitrite precursor therefore for use as a combinedpreparation in the treatment of a disease or medical condition definedabove.

According to a seventh aspect of the present invention there is provideda permeable membrane comprising a pharmaceutically active agent, apharmacologically acceptable acidifying agent and a pharmacologicallyacceptable source of nitrite ions or a nitrite precursor therefore. Themembrane may be fully-, or partially-permeable, includingsemi-permeable, to the passage of nitric oxide. Such membranes canprevent direct contact of the composition with the skin but can permitdiffusion of nitric oxides into the skin. This is particularlyadvantageous in the treatment of areas of broken skin, open wounds orserious burns. In this way the integrity of the wound area is preserved.Suitable membranes include, but are not limited to, polymeric materialssuch as nitrocellulose, cellulose, agarose, polyethylene, polyester (forexample hydrophilic polyester block copolymer) etc. A suitable membranethat can be used in practice in Sympatex™ which is composed of fibres ofhydrophilic polyester block copolymer.

Preferred features for the second and subsequent aspects of theinvention are as for the first aspect mutatis tnutandis.

The invention will now be described, by way of illustration only withreference to the following examples and figures, which are provided forthe purposes of illustration and are not to be construed as beinglimiting on the invention.

FIG. 1 shows the effect of direct application and subsequent removal ofthe treatment on the microcirculatory blood flow in forearm skin andfinger pulps of healthy subjects. The vertical axes are blood flow,photoplethysmography (PPG) relating to microcirculatory volume and laserDoppler fluximetry (LDF) which relates relating to microcirculatory flux(red blood cell count×velocity). The horizontal axis is the time inminutes; NS=not significant; points shown represent the mean value;error bars are 95% confidence; *=p<0.05; **=p<0.01; ***=p<0.001;T=application of gel, and ↓=removal of gel.

FIG. 2 shows the effect of direct application and subsequent removal ofthe treatment on the microcirculatory blood flow in forearm skin andfinger pulps of subjects with severe Raynaud's phenomenon. The verticalaxes are blood flow, photoplethysmography (PPG) relating tomicrocirculatory volume and laser Doppler fluximetry (LDF) which relatesto microcirculatory flux. The horizontal axis is the time in minutes.

FIG. 3 shows nitric oxide diffusion through a selection of membraneswhere the vertical axis shows nitric oxide concentration and thehorizontal axis in the time in minutes. FIG. 3 a shows the results usingSaranwrap™ (SW-01) and FIG. 3 b shows the results using Clingfilm(CF-02).

FIG. 4 shows the diffusion effect of the treatment through a membrane onthe forearm skin microcirculatory blood flow in a healthy subject. Thevertical axis is blood flow, photoplethysmography (PPG) relating tomicrocirculatory volume and the horizontal axis is the time in minutes.

FIG. 5 shows the diffusion effect of the treatment through a membrane onforearm skin microcirculatory blood flow in a healthy subject. Thevertical axis is blood flow, laser Doppler fluximetry (LDF) relating tomicrocirculatory flux and the horizontal axis is the time in minutes.

FIGS. 6 (a)-(i) show the transmembrane diffusion for sodium nitrite andascorbic acid in 0.8% agar gel, using 1% sodium chloride as anintermediate at final concentrations of 500 mM, 250 mM, 165 mM, 50 mM,25 mM, 5 mM, 2.5 mM and 0.5 mM. A control of nitrite and 0.8% agar gelusing 1% sodium chloride as an intermediate was also used. The figureillustrates nitric oxide diffusion through Sympatex™ 10 μm (Akzo Nobel)membrane where the vertical axis shows the nitric oxide concentration inparts per million (PPM) and the horizontal axis shows the time inminutes. In FIGS. 6( a) and 6(b) the initial peaks are artificiallyflattened due to the full scale deflection of the detection device.

FIG. 7 shows the results of the application of nitric oxide generatinggel consisting of 330 mM of sodium nitrite and ascorbic acid in KYJelly™ to the forearm skin and simultaneously to Sympatex™ 10 μmmembrane (Akzo Nobel), which was then applied to the forearm skin of thecontralateral limb if nine healthy subjects. Conditions and experimentalmethods were the same as used for the application of the NO-generationgel on healthy subjects in FIGS. 1, 2, 4 and 5. The vertical axis showsLaser Doppler Fluxmetry units and the horizontal axis shows the time inminutes.

FIG. 8 shows results of pain levels experienced by subjects using VerbalRating Score (VRS); values are categories (percentage); n=100; P<0.0001[1] no pain; [2] minimal sensation; [3] mild pain; [4] moderate pain;[5] severe pain (including withdrawal of hand).

FIG. 9 shows results of Visual Analogue Score (VAS); values are mean±95%CI; n=100; P<0.001.

EXAMPLE 1 Microcirculatory Response to Topical Application ofNo-Generating Gel in Healthy Subjects

A nitric oxide-generating gel (NO-generating gel) was prepared asfollows. Sodium nitrite (Analar™ grade from Sigma, Poole, Dorset, UK)was added to KY Jelly™ (Johnson & Johnson) to make a 5% w/w solution.Ascorbic acid (Sigma) was also added to KY Jelly™ (Johnson & Johnson) tomake a 5% w/w solution. Approximately 0.5 ml of each solution was mixedtogether on the skin of a patient using a sterile swab. When the twosolutions are brought into contact, the ensuing reaction leads to thegeneration of nitric oxide. The reaction may be stopped by cleaning theskin with paper or a swab soaked in ethyl alcohol.

With reference to FIG. 1 the microcirculatory response to topicalapplication of NO-generating gel was measured in 10 healthy subjects.The effect of placebo treatment was measured simultaneously on thecontra-lateral limb. The skin microcirculatory volume was measured byinfra-red photoplethysmography [PPG] and microcirculatory velocity bylaser Doppler fluxmetry [LDF]. All examinations were performed in aquiet, draught-free, temperature and humidity controlled laboratory (24°C.±1° C.; relative humidity 30-40%) in the morning at approximately thesame time of day for each subject.

Placebo treatment did not have any effect upon microcirculatory bloodflow in either the forearm or the finger of the normal subjects. Thevasodilator response to the active treatment reached a plateau phase inall patients within the ten minutes of active gel application. Forearmskin and finger pulp blood flow increased markedly following topicalapplication of a NO-generating gel in the healthy volunteers.

When the active gel was applied to the forearm skin all subjects showeda large vasodilator response to active gel treatment in both volume andflux. This increase in blood flow was sustained after removal of theactive gel. The active gel had no significant effect on fingermicrocirculatory volume (PPG) (FIG. 1: Finger pulp), howevermicrocirculatory flux increased significantly (p<0.01) and remained soafter removal (p<0.01; FIG. 1: Finger pulp).

EXAMPLE 2 Microcirculatory Response to Topical Application ofNO-Generating Gel in Patients with Severe Primary Vasospasm

FIG. 2 shows the microcirculatory response to topical application ofNO-generating gel was measured in 20 patients with severe primaryvasospasm. The effect of the placebo treatment was measuredsimultaneously on the contra-lateral limb. Conditions were the same asthose used for the application of the treatment on healthy subjects inFIG. 1. The skin microcirculatory volume was measured by infra-redphotoplethysmography [PPG] and microcirculatory velocity by laserDoppler fluxmetry [LDF].

Placebo treatment did not have any effect upon microcirculatory bloodflow in either the forearm or the finger of any patients. Thevasodilator response to the active treatment reached a plateau phase inall patients within ten minutes of the application of active gel. Whenthe gel was applied to the forearm skin all patients showed a largevasodilator response to active gel treatment in both volume and flux.This increase in blood flow was sustained after removal of the activegel in both groups (FIG. 2: Forearm and finger pulp). The active gel tothe finger pulp caused a significant increase in microcirculatory volume(p<0.05) which returned rapidly to the resting level on removal of thegel. Active gel also significantly increased finger microcirculatoryflux (p<0.01) which achieved normal values. This increase was sustained,although reduced, after removal of the gel (p<0.05).

EXAMPLE 3 Generation of Nitric Oxide Derived Through a Membrane

FIG. 3 shows the generation of nitric oxide derived from the reactionpreviously detailed through a membrane. Nitric oxide concentrations weremeasured by a nitric oxide sensitive meter: Model 42C ChemiluminescenceNO—NO₂—NO_(x) analyser (Thermo Environmental Instruments Inc., MA USA)connected to a data acquisition system and IBM computer. Measurementswere made continually and readings were taken every 10 seconds for 275minutes. Material 1 was domestic Clingfilm, Material 2 was Saranwrap™(Sigma) and Material 3 was (Sympatex™, Akzo Nobel).

EXAMPLE 4 Microcirculatory Response of the Application of No-GeneratingGel to Three Differing Membrane Materials

FIG. 4 shows the microcirculatory response of the application ofNO-generating gel to three differing membranes which were then appliedto the forearm skin of a healthy subject. Conditions were the same asthose used for the application of the treatment upon healthy subjects inFIG. 1. The skin microcirculatory volume was measured by infra-redphotoplethysmography [PPG]. Material 1 was domestic Clingfilm, Material2 was Saranwrap™ (Sigma) and Material 3 was (Sympatex™, Akzo Nobel).

The increase in microcirculatory blood volume is a reflection of thediffusion of nitric oxide through the membrane towards the skin. Thetransfer of nitric oxide through the membrane is a reflection of thephysical characteristics of the material and is highly variable.Material number 3 (Sympatex™, Akzo Nobel) had a superior diffusionprofile.

EXAMPLE 5 Microcirculatory response of the application of NO-generatinggel to three differing membrane materials

FIG. 5 shows the microcirculatory response of the application ofNO-generating gel to three differing membranes which were then appliedto the forearm skin of a healthy subject. Conditions were the same asthose used for the application of the treatment on healthy subjects inFIG. 1. The skin microcirculatory velocity was measured by laser Dopplerfluxmetry [LDF].

The increase in microcirculatory velocity is a reflection of thediffusion of nitric oxide through the membrane towards the skin. Thetransfer the nitric oxide through the membrane is a reflection of thephysical characteristics of the material and is highly variable.Material number 3 (Sympatex™, Akzo Nobel) had a superior diffusionprofile.

EXAMPLE 6 Comparison of Nitric Oxide Generation Through a Membrane

FIG. 6 shows the generation of nitric oxide derived from the reactiondescribed above through a 10 μm Sympatex™ membrane. Nitric oxideconcentrations were measured by a nitric oxide sensitive meter: Model42C chemiluminescence NO—NO₂—NO_(x) analyser (Thermo EnvironmentalInstrumental Inc., MA, USA) connected to a data acquisition system andan IBM computer. Measurements were made continually and readings weretaken every 10 seconds for 1350 minutes.

The results shown in FIG. 6 illustrate that the transmembrane diffusioncoefficient is closely related to the production of nitric oxide, whichis a direct product of the concentration of both the source of thenitrite ions and the acidifying agent. Furthermore, the resultsdemonstrate that a basal production of nitric oxide is sustained for asignificant period of time after mixing the reagents.

EXAMPLE 7 Microcirculatory Response of the Application of No-GeneratingGel

The nitric oxide generating gel consisting of 330 mM of both sodiumnitrite and ascorbic acid in KY Jelly™ was applied directly to theforearm skin and simultaneously to Sympatex™ 10 μm membrane (AkzoNobel), which was then applied to the forearm skin of the contralaterallimb if nine healthy subjects. Conditions and experimental methods werethe same as used for the application of the NO-generation gel on healthysubjects in FIGS. 1, 2, 4 and 5. The results are shown in FIG. 7. Itshould be noted that in FIG. 7 that the concentrations of the admixtureare in a different unit form (i.e. mM instead of % w/w). Laser DopplerFluxmetry (LDF) measured the skin microcirculatory flux.

The statistically significant increase in microcirculatory flux frombaseline was a reflection of the diffusion of nitric oxide through themembrane towards the skin. This vasodilation, indicated by LDF throughthe membrane ranged from 60-75% (mean 64%) of that observed when theNO-generation gel was applied directly to the skin of the forearm. Theresults shown in FIG. 7 support the observations described in FIG. 1which show that the vasodilator response to the direct treatment reacheda plateau phase in all patients within 10 minutes of gel application. Aplateau phase, although reduced in amplitude was achieved within 16minutes when the NO-generation gel was applied to the membrane andreflects a lag phase which is related to membrane diffusioncharacteristics.

EXAMPLE 8 Use of a Combined Percutaneous Local Anaesthetic andNo-Generating System for Venepuncture

The study was a placebo-controlled double blind trial. The effects ofthe active and placebo treatment were measured at the same time, appliedto different hands. The pain response to cannulation of a dorsal handvein with a 200 Butterfly™ needle was assessed in one hundred healthyvolunteers. The nitric oxide generating system was prepared by mixingtwo viscous gels. The first was a solution of KY Jelly™ and sodiumnitrite (10% w/v) and the second KY Jelly™ and ascorbic acid (10% w/v).This NO-generation gel was termed the placebo treatment, and whencombined with lignocaine in aqueous cream to produce a final 5%anaesthetic concentration, active treatment. Approximately 2 ml of thegel mixtures were separately applied to the skin of the dorsum of thehands (3 cm²) for ten minutes. Following successful cannulation painperception was measured with a verbal rating score (VRS) and a visualanalogue score (VAS).

Pilot studies of 2.5-5.0% xylocalne combined with the NO-generating gelapplied to the ventral surface of the forearm and the dorsal surface ofthe hand suggested a significant level of local anaesthesia was achievedwithin 5-10 minutes as assessed by pin-prick and thermal sensitivitytesting. Furthermore, application of xyolcaine directly to the skinfailed to produce any discernible level of anaesthesia within 20minutes. The anaesthetic used in these initial studies was xylocalne asit was readily available in a pharmaceutical form for mixing.

The aim of this study was to assess how the nitric oxide generatingsystem previously investigated could be combined with lignocaine todecrease the time of onset and increase the effectiveness ofpercutaneous anaesthesia?

Materials and Methods Subjects

The study was a placebo-controlled double blind controlled trial. Onehundred healthy, normotensive volunteers were recruited. A medicalhistory was taken including past medical illness, allergies, smoking,alcohol and consumption of other medically active substances. In aphysical examination blood pressure, pulse rate and rhythm and signs ofdrug and alcohol abuse were also recorded.

Exclusion criteria included: analgesia within preceding 24-hour period;known hypersensitivity to anaesthetics; a history of drug allergy,eczema or psoriasis or with cracks or ulceration of the skin near thevenepuncture/cannulation site; any significant concomitant disease;pregnancy or breast feeding; volunteers taking any medication with knownor potential activity on the cardiovascular system or on blood rheology(for example aspirin or any other NSAID) and blood pressure>160 mmHgsystolic or >100 mmHg diastolic.

The study was approved by the East London and City Health AuthorityEthics Committee [ELCHA]. Participants were admitted to theinvestigation having been provided with a verbal and written explanationand signed a consent form.

Methods

The nitric oxide generating system was prepared by mixing two viscoussolutions (A and B). Solution A was prepared in KY Jelly™ [Johnson &Johnson Ltd.] a sterile lubricant, to which Analar™ grade sodium nitriteto make a 10% (w/v) gel in a sterile plastic specimen pot. Solution Bwas prepared by adding Analar grade ascorbic acid (vitamin C) to KYJelly™ to make a 10% (w/v) gel in a separate sterile plastic pot.

The NO-generation gel was termed the placebo treatment, and when furthersupplemented with lignocaine in aqueous cream to produce a final 5%anaesthetic concentration, active treatment. The NO-generating gel wasused as a placebo treatment because topical application of this systemresults in a pronounced erythema, which would have prevented effectivedouble blinding of the study. Fresh preparations of gels were preparedfor each volunteer. Small quantities (approximately 2.0 ml containing 50milligrams each of sodium nitrite and ascorbic acid) of active andplacebo gel were separately applied to the dorsum of each hand over anarea of 3 cm² and then mixed with a clean cotton bud. Randomisation wasperformed by computer generated allocation. The active treatment wasapplied to the dorsal surface of a randomly selected hand and theplacebo treatment was simultaneously applied to the contralateral hand.Following 10 minutes of application both hands were cleaned prior tovenepuncture.

A vein on each hand within the treatment area was then cannulated usinga 20G butterfly needle, performed in accordance with guidelines detailedin the Royal Hospitals NHS Trust Code of Practice for Phlebotomy withreference to sterility, the risks of infection and contamination. Theleft hand was cannulated first, followed by the right. The success ofcannulation will be recorded by the ability to withdrawal 1 ml of venousblood. If blood was not obtained on the first attempt, then this wascounted as a failed procedure and the patient excluded from the study.Following bilateral cannulation, each hand was be cleaned and dressedappropriately.

Efficacy Measurements

Pain perception is subjective and difficult to measure, hence twooutcome measures were used. The verbal rating score (VRS) and the visualanalogue score (VAS) are well validated criteria (Bradley L. A., ArthrCare Res, 178484 (1993); Woolfson et al Br J Clin Pharmacol 30 273-239(1990)). Each assessment was made using a separate report form withoutvisual reference to previous responses

Following successful bilateral cannulation, a VRS pain classificationwas used with reference first to the left hand, and than repeated forthe right hand. The volunteer was asked the following question: “Howstrong was the pain of the procedure?” and provided with a choice offive answers: [1] No pain; [2] Minimal sensation; [3] Mild pain; [4]Moderate pain; [5] Severe pain (including withdrawal of hand). Thevolunteer selected one answer for each hand by circling the number.

Severity of pain was also assessed by a VAS, consisting of a 100 mmhorizontal line with endpoints that are anchored by descriptors ‘NoPain’ and ‘Severe Pain’. The VAS used with reference first to the lefthand, and than the right hand. For each hand the Volunteer was asked thefollowing question “What did the procedure feel like?” and then wererequested to make a vertical line across the tramline, which representedthe intensity or unpleasantness of his or her pain experienced by theprocedure. Values were measured in millimetres from the left of thetramline.

The application of the gels to the volunteer, bilateral cannulation ofthe dorsal hand veins, and data recording were each performed blindly byseparate investigators.

Study Design and Statistical Analysis

The number of patients required to obtain statistical power wasdifficult to calculate because of the lack of previous studies of thissystem. However, the uncontrolled pilot study allowed a preliminarypower calculations and together with a literature search of similarinvestigations indicated that one hundred subjects would have an 80%power to detect a difference of 25% in the primary outcome measures atp<0.05. Additionally, assessment of one hundred subjects would reducethe influence of any variability of cannulation procedure.

All volunteers received both active and placebo treatmentsimultaneously. All analyses and summaries were performed usingMicrosoft Excel 5.0a and SPSS 6.1.3 commercially available statisticalanalysis packages. Comparisons were made between active and placebotreatment. The Verbal Rating Score was categorical/ordinal data, thusnon-parametric analysis was used (Fisher's Exact test—an extension ofMcNemar's test). The Visual Analogue Score data was an interval scaleand showed normal distribution as confirmed by Ryan-Joiner probabilityplots. Therefore parametric analysis was performed using the 2-samplet-test. A P-value of less than 5% was taken to represent statisticalsignificance.

Results

One hundred healthy volunteers were recruited to the study. Thedemographic data are summarised in Table 1. Additionally, of the onehundred subjects 83 were white European, 8 African/Caribbean, 7 Asian,and 2 Other (Turkish). Forty-four volunteers smoked an average of 5cigarettes per day (range 1-30) and a further eight had ceased smokingfor greater that one month. Eighty-seven volunteers consumed an averageof 14 units per week of alcohol (range 1-60 units).

The cannulation procedures were successfully completed at the firstattempt for all one hundred volunteers. Tolerance and compliance washigh for all subjects. There were no cases of hypersensitivity to eitherpreparation nor adverse event to the investigation.

The verbal rating score (VRS) pain classification recorded significantdifferences in median scores (FIG. 1). The active treatment(lignocaine+NO-generation system) resulted in a reduced pain response tocannulation than the placebo treatment (NO-generation system alone)(p<0.001). However, seven subjects recorded category [4] Moderate painfor the active treatment representing failure of anaesthesia. Median VRSwere similar between the sexes for active and placebo treatments.

The visual analogue score (VAS) were also significantly differentbetween the two groups (FIG. 2). The active treatment resulted insignificantly less response to cannulation than the placebo treatment(p<0.001). The active formulation produced a reduction in mean VAS painscore of 40.3%. There were no differences between male and female meanVAS pain scores for either treatment.

TABLE 1 Volunteer demographics mean ± S.D Parameters (range) median sex56:44 (Male:Female) age (years) 27.6 ± 5.8  27.0 (20−40) BMI 25.1 ± 4.4 24.5 (weight/height²) (17.5−42.0) MAP 89.2 ± 10.0 88.7  (69.7−118.7)Heart rate   74 ± 11.5 74 (bpm)  (54−111)

Mean arterial blood pressure [MAP] was calculated as:

$\frac{{{Systolic}\mspace{14mu} {blood}\mspace{14mu} {pressure}} - {{Diastolic}\mspace{14mu} {blood}\mspace{14mu} {pressure}}}{3} + {{Diastolic}\mspace{14mu} {blood}\mspace{14mu} {pressure}}$

Findings

Topical application of the NO-generating gel and lignocaine mixturesignificantly reduced the pain associated with venous cannulation. Theformulation resulted in a decreased VRS<0.0001) and produced a reductionin mean VAS of >40% compared to the placebo gel (P<0.001).

Interpretation

This investigation suggests that topical application for ten minutes ofthe combination of anaesthetic with a nitric oxide-generation system mayprovided effective anaesthesia for venous cannulation in adults. Noadverse effects were reported with this treatment.

Discussion

This study suggests that a ten-minute topical application of thecombination of lignocaine with a nitric oxide-generation system mayprovide effective anaesthesia for venous cannulation of the dorsum ofthe hand in adults. These findings are important as cannulation of thedorsal hand vein is commonly described as a painful procedure incomparison to other anatomical regions. The degree of anaesthesiaobserved was achieved following only ten minutes of application. This isnot the case for existing commercially available treatments.

The main route for a drug molecule penetrating the stratum cornuem isthrough the intercellular matrix while very limited drug penetrationoccurs via the intracellular corneocytes (Singh S, and Singh J. Med ResRev 13(5), pages 569-621 (1993)). Additionally, drugs may enter the skinthrough specialised structures such as sweat ducts and hair follicles.The influence of the NO-generation system on the route of lignocainetransmission is unclear at this time and awaits elaboration, but may berelated to increased cutaneous blood flow.

Although other investigators using EMLA™ and Ametop Gel™ report variablelevels of erythema dependent upon the duration of application(Arrowsmith J, and Campbell C. Arch Dis Child 82(4) pages 309-310(2000)), clinical experience suggests that they do significantly effectthe tone of the venous vessels. The nitric oxide component of thissystem results in an increase in luminal diameter (consequent tovasodilatation) and the apparent attenuation of vasospasm may assist inthe cannulation of small, vasospastic or friable vessels.

A further observation in the treatment area with an associated increasein the colour contrast between the blue venous vessels and the rederythema of the skin. The delineated area of gel application facilitatedthe identification of the region of treatment and targeting of the veinby the clinician. The erythema was transient in nature and was notassociated with tissue oedema.

The ideal percutaneous local anaesthetic preparation will need to fulfila number of requirements [a] To profoundly anaesthetise the skin surfaceand underlying tissues; [b] Have a more rapid onset of action; [c]Increase vasodilation of venous vessels; [d] Have a prolonged durationof action; [e] Contain the minimum necessary concentration of localanaesthetic; [f] Produce no systemic toxicity; [g] Produce nosignificant local reactions; [h] Avoids sensitisation to future skinapplication. The formulation described in this study appears to have thepotential to fulfil these criteria.

This investigation is a preliminary report. However, the findings ofthis study suggest that future studies are required to investigate theeffects of anaesthetic type, formulation and concentration, duration ofaction and penetration depth, anatomical and physiological variation andcomparisons with both EMLA™ and Ametop Gel™. A further interestingpossibility exists that the nitric oxide-generation gel may not be atrue placebo and may in fact have some degree of anaesthetic effect(Redford et al Brain 120(12), pages 2149-2157 (1997); Sousa A M, andPrado W A., Brain Res 897(1-2), pages 9-19 (2001)). However, ifdemonstrated this effect would clearly add to the efficacy of thecombined system as a topically-applied anaesthesia.

In summary, this investigation is believed to be the first to describethe addition of a primary pharmaceutical agent with a topically-appliednitric oxide generation system. The combined system may overcome thelimitations associated with conventional transdermal drug applicationand be developed into a clinically useful transdermal deliverytechnology for a broad spectrum of pharmaceutical agents.

1-20. (canceled)
 21. A membrane associated with a pharmaceuticallyactive agent, a pharmacologically acceptable acidifying agent, and apharmacologically acceptable source of nitrite ions or a nitriteprecursor therefor.
 22. The membrane of claim 21, wherein thepharmacologically acceptable source of nitrite ions is an alkali metalnitrite or an alkaline earth metal nitrite.
 23. The membrane of claim21, wherein the pharmacologically acceptable acidifying agent is anorganic acid.
 24. The membrane of claim 23, wherein the organic acid ischosen from ascorbic acid, salicylic acid, acetyl salicylic acid, a(C₁-C₆) alkyl carboxylic acid, and citric acid, or a salt or derivativethereof.
 25. The membrane of claim 21, wherein the pharmaceuticallyactive agent is chosen from an anaesthetic, an analgesic, a hormone, animmunosuppressant drug, and a steroid.
 26. The membrane of claim 25,wherein the pharmaceutically active agent is an anaesthetic chosen fromamethocaine (tetracaine), lignocaine (lidocaine), xylocalne,bupivacaine, prilocalne, ropivacaine, benzocaine, mepivocaine, andcocaine, or any mixture thereof.
 27. The membrane of claim 21, whereinthe final nitrite ion concentration is up to 20%.
 28. The membrane ofclaim 21, wherein the membrane comprises a polymeric material.
 29. Themembrane of claim 28, wherein the polymeric material is chosen fromnitrocellulose, cellulose, agarose, polyethylene, and polyester.
 30. Themembrane of claim 29, wherein the polyester is hydrophilic polyesterblock copolymer.
 31. The membrane of claim 21, wherein the membrane isfully or partially permeable to the passage of nitric oxide.
 32. Themembrane of claim 21, wherein the membrane prevents direct contact ofthe pharmacologically acceptable acidifying agent and thepharmacologically acceptable source of nitrite ions or a nitriteprecursor therefor with the skin but permits diffusion of nitric oxideinto the skin.
 33. A method of treating broken skin, open wounds, orburns in a patient in need thereof comprising applying the membrane ofclaim 32 to the patient on the area of broken skin, open wound, or burn.34. A method of providing pain relief or preventing pain in a patient inneed of localized anaesthesia comprising applying the membrane of claim26 to the patient at a localized painful region or on a region that willbecome painful.
 35. The method of claim 34, wherein the pharmaceuticallyacceptable acidifying agent and the pharmacologically acceptable sourceof nitrite ions are formulated in or on the membrane, further comprisingapplying the membrane on the patient for effecting a topical reaction onthe patient's skin at the site of administration.
 36. The method ofclaim 35, wherein the pharmaceutically active agent is formulated in oron the membrane, or is administered to the patient with the membrane.